Project #1. Impact of HIV-1 on the Rho GTPase-mediated neuronal cell development. It is now well accepted that the HIV-1 regulatory protein, Tat, has a neurotoxic effect and that treatment of both human and rodent neurons with Tat induces apoptosis by mechanisms that are not fully understood. The cellular protein, Pur-alpha, is a key target of Tat for directing the regulation of HIV-1 transcription, translation, and viral RNA transport. Indeed, both Pur-alpha and Tat have been detected in the same cells within HIVE infected lesions. Results in the Pur-alpha knockout mouse model have revealed a critical role for Pur-alpha during development, particularly in the coordinated development and differentiation of neuronal cells throughout the brain. Over the past several years, it has become clear that the RhoGTPases and related molecules play an important role in neuronal cell development, including neurite outgrowth, differentiation, axon pathfinding, dendritic spine formation, as well as neuronal cell maintenance. Results from our studies indicate that Tat has the ability to disturb this pathway by stimulating RhoA activation, an event that is accompanied by conversion of RhoA[unreadable]GDP to RhoA[unreadable]GTP by interacting with guanine nucleotide exchange factor (GEF) family members including PDZ-RhoGEF. Indeed, our preliminary results suggest that Tat may mediate RhoA activity via interaction with PDZ-RhoGEF. Upon its activation, RhoA is usually positioned in the plasma membrane, and via a series of signaling pathways that involve Rock, MAPK, and JNK1, NFkB induces neurite retraction. Moreover, activation of RhoA via heteromeric G-proteins including Ga12 and G<x13 can stimulate GSK3(3 which is also known to be involved in HIV-1 neurotoxicity. On the other hand, Tat can block activation of another key RhoGTPase, i.e. Rac1 and its partner p21-activated kinase 1 (PAK1), which appears to regulate actin cytoskeletal dynamics, maintain neuronal cell integrity, and promote neurite outgrowth. In addition, Tat has recently been shown to inhibit this pathway by acting through PAK1 and JNK1. Finally, Rac1 and RhoA were found to display aberrant expression and subcellular localization in the Pur-alpha knockout mouse model, suggesting that Pur-alpha is required for their proper function. Based on these preliminary observations, we hypothesize that Pur-alpha has a central role in the well-balanced activities of RhoA and Rac, and that its genetic ablation or its functional alteration by associating with Tat can derail several key parameters in RhoA and Rac signaling pathways including p115/PDZ and PAK, respectively, and results in pathological features that are commonly seen in neuronal cells of AIDS patients with CNS disorders. In this research project, we will perform a series of cell biological and molecular studies to unravel the molecular basis of Tat cross-talk with Rho GTPase and its impact on neuronal cell integrity.